ONCOLOGY LETTERS 9: 2085-2089, 2015
Expression of CC chemokine receptor 5 in clear cell renal cell carcinoma and its clinical significance KOO HAN YOO1, DONG‑GI LEE1, KYU YEOUN WON2, SUNG‑JIG LIM2, YONG‑KOO PARK2 and SUNG‑GOO CHANG1 Departments of 1Urology and 2Pathology, School of Medicine, Kyung Hee University, Seoul 130‑702, Republic of Korea Received June 27, 2014; Accepted March 4, 2015 DOI: 10.3892/ol.2015.3048 Abstract. DNA hypomethylation was the initial epigenetic abnormality recognized in human malignancy. In the present study, the GoldenGate high‑throughput genotyping assay was adapted to determine the methylation state of 1,505 specific CpG sites in 807 cancer‑related genes. The methylation results revealed that CC chemokine receptor 5 (CCR5) was hypomethylated (mean β ‑value difference, ‑0.21) in clear cell renal cell carcinoma (CCRCC) tissue. Tissue samples from 61 CCRCC cases were used for immunohistochemical staining, and patients with low CCR5 expression (n=44) were compared with those with high CCR5 expression (n=17). Tumor (T) stage was significantly lower in the low expression group compared with the high expression group (P=0.047). The Fuhrman grade of patients in the low expression group was significantly lower than that of patients in the high expression group (P=0.044). Whilst the node (N) and metastasis (M) stages of the CCR5 low expression group appeared to be lower compared with those of the CCR5 high expression group; this difference was not statistically significant (N stage, P= 0.632; M stage, P=0.896). Additionally, patients in the low expression group had lower risks of postoperative tumor recurrence (P=0.110) and mortality from CCRCC (P=0.159) compared with those in the high expression group, however, this was also without statistical significance. The results indicate that CCR5 hypomethylation is associated with cancer tissue to a greater extent than normal tissue. Although the biological function of CCR5 in CCRCC remains to be established, low CCR5 expression is associated with low T stage and low Fuhrman grade in these patients.
Correspondence to: Professor Sung‑Goo Chang, Department of
Urology, School of Medicine, Kyung Hee University, 1 Hoegi‑dong, Dongdaemun‑ku, Seoul 130‑702, Republic of Korea E‑mail: [email protected]
Key words: carcinoma, renal cell, DNA methylation, receptors, CC chemokine receptor 5
Introduction As the most common neoplasm in the kidney, renal cell carcinoma (RCC) comprises 3% of all cases of adult cancer. In the United States, the incidence of RCC is increasing, with an estimated 63,920 new cases and 13,860 mortalities in 2014 (1). In South Korea, RCC accounts for ~1% of all primary malignancies and is the tenth most common cancer in males (2). RCC may be classified into a number of subtypes, including papillary, chromophobe and collecting duct RCC, clear cell RCC (CCRCC) and other rare subtypes. CCRCC represents 70% of all RCCs and tends to have a poorer prognosis compared with other RCCs (3). Epigenetic changes may initiate cancer and promote progression (4). Epigenetics may be described as a stable alteration in gene expression potential that takes place during cell proliferation and development, without any change in gene sequence (5). In cancer, DNA hypermethylation is critical in gene expression. Methylation in cytosine-phosphateguanine (CpG) islands may inhibit gene expression, and CpG hypermethylation in promoter regions may represent one mechanism of carcinogenesis; this may also provide markers for tumor initiation and progression. Hypomethylation is the second type of methylation defect that is observed in a wide range of malignancies. Hypomethylation involves repeated DNA sequences, including long interspersed nuclear elements (LINEs), whereas hypermethylation involves CpG islands. Chemokines regulate cancer cell migration and contribute to cancer cell proliferation and survival (6). Approximately 45 chemokines and 20 chemokine receptors have been identified, and may be grouped into four categories: C, CC, CXC and CX3C (7). Chemokine receptors relay their signals through heterotrimeric guanine nucleotide‑binding proteins (G proteins) (7). CC chemokine receptor 5 (CCR5) belongs to the trimeric G protein‑coupled seven‑transmembrane domain receptor superfamily (9), and binds to the RANTES (CCL5), macrophage inflammatory protein (MIP)‑1α (CCL3), and MIP‑1β (CCL4) chemokines (10). CCR5 is involved in the chemotaxis of leukocytes to sites of inflammation, and is important in the recruitment of macrophages, T cells and monocytes (11). In the present study, the methylation profile of CCR5 was investigated in CCRCC. The association between tumoral CCR5 immunohistochemical expression and clinicopathological parameters was also evaluated.
2086
YOO et al: CCR5 EXPRESSION IN CCRCC
Figure 1. Low magnification view showing high CCR5 expression in the core of clear renal cell carcinoma on the left and low CCR5 expression in the core on the right (magnification, x20). CCR5, CC chemokine receptor 5.
Materials and methods Patients and preparation of DNA samples. The GoldenGate high‑throughput genotyping assay was adapted to determine the methylation state of 1,505 specific CpG sites in 807 cancer‑related genes (12). Tissue specimens consisted of 62 cancer tissues and 62 matched adjacent normal tissues from CCRCC patients at Kyung Hee University Hospital (Seoul, South Korea). The Institutional Review Board of Kyung Hee University Hospital approved this study (KHNMC IRB 2013‑040). DNA was extracted as previously described (13). Methylation profiling and validation. Bisulfite conversion of all DNA samples was performed with the EZ‑96 DNA Methylation™ kit (Zymo Research, Orange, CA, USA) according to the manufacturer's instructions. Following bisulfite treatment, the methylcytosine content was quantified using Illumina's GoldenGate Methylation Cancer Panel I microarray (Illumina Inc., San Diego, CA, USA) (12). The raw methylation ratios were calculated using the Methylation Module in Illumina's BeadStudio following normalization to a background level derived by averaging the signals of an internal negative control. The methylation status of the CpG sites was examined by bisulfite sequencing. The procedure described previously by Herman et al (14) was adopted, with slight modification. Tissue microarray and immunohistochemistry. For immunohistochemical staining, tissue samples from 61 CCRCC cases were used. All neoplasms were surgically resected at Kyung Hee University Hospital between 2006 and 2013. The tissue microarrays were assembled using a commercially available manual tissue microarrayer (Quick‑Ray; Unitma Co., Ltd., Seoul, South Korea). Three representative tumor cores with diameters of 2.0 mm were punched from each tumor tissue block. Each of the tissue microarray blocks contained three normal kidney tissue cores (Fig. 1). Immunohistochemistry was performed on 4‑µm tissue sections from each tissue microarray block using the Bond Polymer Intense Detection System (Vision BioSystems, Victoria, Australia). Sections were incubated for 15 min at ambient temperature with primary rabbit polyclonal antibodies to CCR5 (dilution, 1:100; Novus Biologicals, Cambridge, UK), using a biotin‑free polymeric horseradish peroxidase‑linked antibody conjugate system
in a Bond‑max automatic slide stainer (Vision BioSystems). Nuclei were counterstained with hematoxylin. The negative control was treated in an identical manner using mouse immunoglobulin G instead of primary antibody. The degree of expression based on immunohistochemistry was classified by three pathologists. Semiquantitative analysis of immunoreactivity was performed according to intensity and proportion: The intensity score was as follows: 0, no staining; 1, weak but detectable staining; 2, distinct staining; or 3, strong staining. The proportion score was as follows: 0, 0% stained cells; 1, 1‑33% stained cells; 2, 34‑66% stained cells; or 3, 67‑100% stained cells. These two scores were multiplied together for a total score, categorized as follows: 0‑4, low expression; and 5‑9, high expression (Fig. 2). Statistical analysis. Statistical analyses were performed using SPSS software (version 15.0; SPSS, Inc., Chicago, IL, USA). A χ2 test and linear‑by‑linear association were used to evaluate the association of the degree of expression by immunohistochemistry with clinicopathological variables. The overall survival was defined as the time interval between the primary radical or partial nephrectomy and the last follow‑up or mortality. The recurrence‑free survival period was defined as the time interval between the primary radical or partial nephrectomy and the last follow‑up or evidence of recurrence. Survival was estimated using the Kaplan‑Meier method. All statistical tests were two sided, and P
Expression of CC chemokine receptor 5 in clear cell renal cell carcinoma and its clinical significance KOO HAN YOO1, DONG‑GI LEE1, KYU YEOUN WON2, SUNG‑JIG LIM2, YONG‑KOO PARK2 and SUNG‑GOO CHANG1 Departments of 1Urology and 2Pathology, School of Medicine, Kyung Hee University, Seoul 130‑702, Republic of Korea Received June 27, 2014; Accepted March 4, 2015 DOI: 10.3892/ol.2015.3048 Abstract. DNA hypomethylation was the initial epigenetic abnormality recognized in human malignancy. In the present study, the GoldenGate high‑throughput genotyping assay was adapted to determine the methylation state of 1,505 specific CpG sites in 807 cancer‑related genes. The methylation results revealed that CC chemokine receptor 5 (CCR5) was hypomethylated (mean β ‑value difference, ‑0.21) in clear cell renal cell carcinoma (CCRCC) tissue. Tissue samples from 61 CCRCC cases were used for immunohistochemical staining, and patients with low CCR5 expression (n=44) were compared with those with high CCR5 expression (n=17). Tumor (T) stage was significantly lower in the low expression group compared with the high expression group (P=0.047). The Fuhrman grade of patients in the low expression group was significantly lower than that of patients in the high expression group (P=0.044). Whilst the node (N) and metastasis (M) stages of the CCR5 low expression group appeared to be lower compared with those of the CCR5 high expression group; this difference was not statistically significant (N stage, P= 0.632; M stage, P=0.896). Additionally, patients in the low expression group had lower risks of postoperative tumor recurrence (P=0.110) and mortality from CCRCC (P=0.159) compared with those in the high expression group, however, this was also without statistical significance. The results indicate that CCR5 hypomethylation is associated with cancer tissue to a greater extent than normal tissue. Although the biological function of CCR5 in CCRCC remains to be established, low CCR5 expression is associated with low T stage and low Fuhrman grade in these patients.
Correspondence to: Professor Sung‑Goo Chang, Department of
Urology, School of Medicine, Kyung Hee University, 1 Hoegi‑dong, Dongdaemun‑ku, Seoul 130‑702, Republic of Korea E‑mail: [email protected]
Key words: carcinoma, renal cell, DNA methylation, receptors, CC chemokine receptor 5
Introduction As the most common neoplasm in the kidney, renal cell carcinoma (RCC) comprises 3% of all cases of adult cancer. In the United States, the incidence of RCC is increasing, with an estimated 63,920 new cases and 13,860 mortalities in 2014 (1). In South Korea, RCC accounts for ~1% of all primary malignancies and is the tenth most common cancer in males (2). RCC may be classified into a number of subtypes, including papillary, chromophobe and collecting duct RCC, clear cell RCC (CCRCC) and other rare subtypes. CCRCC represents 70% of all RCCs and tends to have a poorer prognosis compared with other RCCs (3). Epigenetic changes may initiate cancer and promote progression (4). Epigenetics may be described as a stable alteration in gene expression potential that takes place during cell proliferation and development, without any change in gene sequence (5). In cancer, DNA hypermethylation is critical in gene expression. Methylation in cytosine-phosphateguanine (CpG) islands may inhibit gene expression, and CpG hypermethylation in promoter regions may represent one mechanism of carcinogenesis; this may also provide markers for tumor initiation and progression. Hypomethylation is the second type of methylation defect that is observed in a wide range of malignancies. Hypomethylation involves repeated DNA sequences, including long interspersed nuclear elements (LINEs), whereas hypermethylation involves CpG islands. Chemokines regulate cancer cell migration and contribute to cancer cell proliferation and survival (6). Approximately 45 chemokines and 20 chemokine receptors have been identified, and may be grouped into four categories: C, CC, CXC and CX3C (7). Chemokine receptors relay their signals through heterotrimeric guanine nucleotide‑binding proteins (G proteins) (7). CC chemokine receptor 5 (CCR5) belongs to the trimeric G protein‑coupled seven‑transmembrane domain receptor superfamily (9), and binds to the RANTES (CCL5), macrophage inflammatory protein (MIP)‑1α (CCL3), and MIP‑1β (CCL4) chemokines (10). CCR5 is involved in the chemotaxis of leukocytes to sites of inflammation, and is important in the recruitment of macrophages, T cells and monocytes (11). In the present study, the methylation profile of CCR5 was investigated in CCRCC. The association between tumoral CCR5 immunohistochemical expression and clinicopathological parameters was also evaluated.
2086
YOO et al: CCR5 EXPRESSION IN CCRCC
Figure 1. Low magnification view showing high CCR5 expression in the core of clear renal cell carcinoma on the left and low CCR5 expression in the core on the right (magnification, x20). CCR5, CC chemokine receptor 5.
Materials and methods Patients and preparation of DNA samples. The GoldenGate high‑throughput genotyping assay was adapted to determine the methylation state of 1,505 specific CpG sites in 807 cancer‑related genes (12). Tissue specimens consisted of 62 cancer tissues and 62 matched adjacent normal tissues from CCRCC patients at Kyung Hee University Hospital (Seoul, South Korea). The Institutional Review Board of Kyung Hee University Hospital approved this study (KHNMC IRB 2013‑040). DNA was extracted as previously described (13). Methylation profiling and validation. Bisulfite conversion of all DNA samples was performed with the EZ‑96 DNA Methylation™ kit (Zymo Research, Orange, CA, USA) according to the manufacturer's instructions. Following bisulfite treatment, the methylcytosine content was quantified using Illumina's GoldenGate Methylation Cancer Panel I microarray (Illumina Inc., San Diego, CA, USA) (12). The raw methylation ratios were calculated using the Methylation Module in Illumina's BeadStudio following normalization to a background level derived by averaging the signals of an internal negative control. The methylation status of the CpG sites was examined by bisulfite sequencing. The procedure described previously by Herman et al (14) was adopted, with slight modification. Tissue microarray and immunohistochemistry. For immunohistochemical staining, tissue samples from 61 CCRCC cases were used. All neoplasms were surgically resected at Kyung Hee University Hospital between 2006 and 2013. The tissue microarrays were assembled using a commercially available manual tissue microarrayer (Quick‑Ray; Unitma Co., Ltd., Seoul, South Korea). Three representative tumor cores with diameters of 2.0 mm were punched from each tumor tissue block. Each of the tissue microarray blocks contained three normal kidney tissue cores (Fig. 1). Immunohistochemistry was performed on 4‑µm tissue sections from each tissue microarray block using the Bond Polymer Intense Detection System (Vision BioSystems, Victoria, Australia). Sections were incubated for 15 min at ambient temperature with primary rabbit polyclonal antibodies to CCR5 (dilution, 1:100; Novus Biologicals, Cambridge, UK), using a biotin‑free polymeric horseradish peroxidase‑linked antibody conjugate system
in a Bond‑max automatic slide stainer (Vision BioSystems). Nuclei were counterstained with hematoxylin. The negative control was treated in an identical manner using mouse immunoglobulin G instead of primary antibody. The degree of expression based on immunohistochemistry was classified by three pathologists. Semiquantitative analysis of immunoreactivity was performed according to intensity and proportion: The intensity score was as follows: 0, no staining; 1, weak but detectable staining; 2, distinct staining; or 3, strong staining. The proportion score was as follows: 0, 0% stained cells; 1, 1‑33% stained cells; 2, 34‑66% stained cells; or 3, 67‑100% stained cells. These two scores were multiplied together for a total score, categorized as follows: 0‑4, low expression; and 5‑9, high expression (Fig. 2). Statistical analysis. Statistical analyses were performed using SPSS software (version 15.0; SPSS, Inc., Chicago, IL, USA). A χ2 test and linear‑by‑linear association were used to evaluate the association of the degree of expression by immunohistochemistry with clinicopathological variables. The overall survival was defined as the time interval between the primary radical or partial nephrectomy and the last follow‑up or mortality. The recurrence‑free survival period was defined as the time interval between the primary radical or partial nephrectomy and the last follow‑up or evidence of recurrence. Survival was estimated using the Kaplan‑Meier method. All statistical tests were two sided, and P
